Pursuit of in vitro biomimetic organ growth has spurred a number of recent investigations of methods to generate three-dimensional (3D) scaffolding structures and techniques for cellular seeding (Schmeichel K L and Bissell M J. (2003) J. Cell Sci. 116:2377-2388; Cukierman et al. (2001) Science 294:1708-1712; Tsang V L and Bhatia S N. (2006) Adv. Biochem. Eng. Biotechnol. 103:189-205). In vitro organs that have demonstrated functionality similar to natural organs have been produced previously by cell re-seeding onto cadaver-derived, de-cellularized protein scaffolds (Ott et al. (2010) Nat. Med. 16:927-933). These results suggest that the development of a sufficiently complex 3D cell scaffold may allow for the re-growth of organs de novo.
Cell scaffolding structures, commonly referred to as extracellular matrices (ECM), should be constructed from benign compounds (Owen S C and Schoichet M S. (2010) Biomed. Mater. Res. A 94:1321-1331). Scaffold materials will either decompose metabolically during cell propagation or be fully incorporated into the final organ. Materials that have been suggested include ceramics, chitosan, collagen, peptides, polyethylene glycol (PEG), polysaccharides, and various synthetic biomaterials (Bartolo et al. (2011) Advances on Modeling in Tissue Engineering. Dordrecht: Springer Netherlands, pp. 137-176). For applications involving human hosts, material selection criteria must consider toxicity, antigenicity, mechanical strength, thermal stability, and porosity.
Collagen has been used frequently in previous investigations of ECM development (Bartolo et al. (2011) Advances on Modeling in Tissue Engineering. Dordrecht: Springer Netherlands, pp. 137-176; Liu et al. (2007) J. Biomed. Mater. Res. Part B 85:519-528; Glowacki J and Mizuno S (2007) Biopolymers 89:338-344). It is a crystalline (Hulmes D J S and Miller A (1979) Nature 282:878-880; Prockop D J and Feralta A (1998) J. Struct. Biol. 122:111-118; Kadler er al. (1996) Biochem. J. 316:1-11), triple helical molecule (Hulmes et al. (1973) J. Molecular Biol. 79:137-148) and a favorable material for biomedical applications, since it is a biodegradable and biocompatible, insoluble fibril with high mechanical strength and relatively low immunogenicity (Hsu et al. (1994) Biorheology 31:21-36; Lynn et al. (2004) J. Biomed. Mater. Res. B Appl. Biomater. 71B:343-354; Zeugolis et al. (2008) Biomaterials 29:2293-2305).
Gelatin is the incompletely denatured form of collagen and comprises variable-length peptides which have fibrillar structure but lack configurational order (Veis et al. (1961) Arch. Biochem. Biophys. 94:20-31). In vivo use of gelatin has been successfully demonstrated by implantation in animal models, with results that suggest low toxicity and reduced antigenicity relative to collagen (Fassina et al. (2010) Conf. Proc. IEEE Eng. Med. Biol. Soc. 2010:247-250; Ponticiello et al. (2000) J. Biomed. Mater. Res. 52:246-255). Furthermore, gelatin is relatively inexpensive compared to collagen and its cell adhesion and proliferation characteristics are essentially indistinguishable (Ratanavaraporn et al. (2006) J. Metals Materials Minerals 16:31-36). Gelatin's use in ECM is complicated by its lack of 3D structural integrity, lower melting temperatures, and rapid dissolution in water (Veis et al. (1961) Arch. Biochem. Biophys. 94:20-31; Sachlos E and Czernuszka J T (2003) Eur. Cell. Mater. 5:29-40). For use as cell scaffolds, recent studies have sought to increase the mechanical and thermal resiliency through compositing with various compounds (Das et al. (2013) Biomacromolecules 14(2):311:321; Hunger et al. (2013) Acta. Biomater. 9(5):6338-6348) and by utilization of covalent cross-linking agents (Huang et al. (2005) Biomaterials 26:7616-7627). Many cross-linking agents, however, are toxic or immunogenic, e.g., glutaraldehyde (Glowacki J and Mizuno S (2007) Biopolymers 89:338-344).
The utilization of sugars as a gelatin cross-linking agent has been previously investigated (Cortesi et al. (1998) Biomaterials 19:1641-1649), and its usefulness in vivo without host toxicity successfully demonstrated. Cross-linking between gelatin and both non-reducing and reducing sugars can be observed without catalysis; however, due to weak ionic interactions, dissolution still occurs at physiological temperatures (lower than 37° C.), albeit at a reduced rate (Cortesi et al. (1998) Biomaterials 19:1641-1649). The formation of covalent interactions is therefore necessary to produce a thermostable compound. The Maillard reaction pathway generates covalent bonds between reducing sugars and protein amine groups (Easa et al. (1996) Int. J. Biol. Macromol. 18:297-301) and produces observable physical changes in gelatin and other protein matrices (Lederer et al. (1998) Bioorg. Med. Chem. 6:993-1002; Nakajima et al. (2008) Biosci. Biotechnol. Biochem. 72:295-302; Su et al. (2011) J. Sci. Food Agric. 91:2457-2462).
Glycation end products are the resultant glycosylated proteins generated by Maillard chemistry (Ohan et al. (2002) J. Biomed. Mater. Res. 60:384-391; Goldin et al. (2006) Circulation 114:597-605). Sugar cross-linking of gelatin molecules has been shown to increase stiffness and decrease solubility (Goldin et al. (2006) Circulation 114:597-605; Tomihata et al. (1994) Polymers of Biological and Biomedical Significance. American Chemical Society. Chapter 24, 275-286). As demonstrated quantitatively herein, ultraviolet (UV) radiation can provide the necessary energetic input required to crosslink gelatin. The cross-linked gelatin product exhibits good thermal stability and has the potential for future 3D cell scaffold application.